A homozygous loss-of-function mutation leading to CYBC1 deficiency causes chronic granulomatous disease

Abstract

Mutations in genes encoding subunits of the phagocyte NADPH oxidase complex are recognized to cause chronic granulomatous disease (CGD), a severe primary immunodeficiency. Here we describe how deficiency of CYBC1, a previously uncharacterized protein in humans (C17orf62), leads to reduced expression of NADPH oxidase's main subunit (gp91^phox) and results in CGD. Analyzing two brothers diagnosed with CGD we identify a homozygous loss-of-function mutation, p.Tyr2Ter, in CYBC1. Imputation of p.Tyr2Ter into 155K chip-genotyped Icelanders reveals six additional homozygotes, all with signs of CGD, manifesting as colitis, rare infections, or a severely impaired PMA-induced neutrophil oxidative burst. Homozygosity for p.Tyr2Ter consequently associates with inflammatory bowel disease (IBD) in Iceland (P = 8.3 × 10^-8; OR = 67.6), as well as reduced height (P = 3.3 × 10^-4; -8.5 cm). Overall, we find that CYBC1 deficiency results in CGD characterized by colitis and a distinct profile of infections indicative of macrophage dysfunction.

Fig. 1

Pedigree and burst test results for the two probands, and the CYBC1 protein. a Pedigree of the two CGD brothers showing their CYBC1 p.Tyr2Ter genotypes. Squares represent males, circles represent females, and a slashed symbol indicates a deceased individual. Filled symbols represent affected individuals; the two affected are referred to as individuals A and B in the manuscript. The genotype of the p.Tyr2Ter mutation (NP_001028218.1:p.Tyr2Ter; NM_001033046.3:c.6C>G; hg38 position chr17:82,449,249) is indicated with M and W, M representing the mutated allele and W the wild type. M/M therefore indicates homozygous status, W/M indicates heterozygous status and W/W a non-carrier. b Neutrophil oxidative burst test for the two CGD brothers homozygous for CYBC1 p.Tyr2Ter (individuals A and B) and their controls, test was performed pre-HSCT. Left panel shows fluorescent peaks for unstimulated and PMA stimulated neutrophils from the controls, and the right panel shows peaks for unstimulated and PMA stimulated neutrophils from the two homozygous brothers. Negative and positive cells are defined by setting a gate for unstimulated cells. Neutrophils from individuals A and B failed to generate an oxidative burst equivalent to their controls, their respective stimulation indices were SI_A = 1.34 and SI_B = 2.50. c Topological prediction of CYBC1 (NP_001028218.1). CYBC1 is predicted to be a transmembrane protein, spanning the lipid bilayer via two transmembrane regions (aa 21–39 and aa 45–63). A red diamond represents the p.Tyr2Ter mutation at the second amino acid of the protein

Fig. 2

Effect of p.Tyr2Ter on CYBC1, the neutrophil oxidative burst, and gp91^phox. a CYBC1 protein expression, relative to ACTIN, in lymphocytes from CYBC1 p.Tyr2Ter homozygous individuals D and F (triangles), four heterozygous carriers (squares), and matched (age and sex) non-carriers (circles). CYBC1 expression was not detected in the homozygous individuals in contrast to their matched non-carriers, and was reduced by 53% in the heterozygous carriers, compared to matched non-carriers. The analysis was performed by western blot (shown in Supplementary Fig. 3 and 4), where ACTIN was used as a loading control. b Neutrophil oxidative burst test for CYBC1 p.Tyr2Ter homozygous individual D and her matched (age and sex) non-carrier. Left panel shows fluorescent peaks for unstimulated and PMA stimulated neutrophils from the non-carrier, and the right panel shows peaks for unstimulated and PMA stimulated neutrophils from homozygous individual D. Negative and positive cells are defined by setting a gate for unstimulated cells. Neutrophils from individual D failed to generate an oxidative burst equivalent to her matched non-carrier, the stimulation index for homozygous individual D was SI_D = 2.35. c Protein expression of gp91^phox, relative to ACTIN, in monocyte-derived macrophages from CYBC1 p.Tyr2Ter homozygotes D and F (squares) and their matched (age and sex) non-carriers (circles). gp91^phox expression was absent in the two homozygotes in contrast to their matched non-carriers. The analysis was performed by western blot (shown in Supplementary Fig. 5), where ACTIN was used as a loading control. d CYBC1 and gp91^phox expression in fresh neutrophils from CYBC1 p.Tyr2Ter homozygous individual D and her matched (age and sex) non-carrier. CYBC1 expression was not detected in the homozygous individual in contrast to the non-carrier. gp91^phox expression was ~50% lower in the homozygote than her matched non-carrier. The analysis was performed by western blot, and ACTIN was used as a loading control